1. Field of the Invention
The invention relates to dopamine receptors and the genes corresponding to such receptors. In particular, it relates to a synthetic dopamine receptor having drug dissociation properties similar to those properties characteristic of the human dopamine receptor D4.
2. Background
Dopamine is a neurotransmitter that participates in a variety of different functions mediated by the nervous system, including vision, movement, and behavior. See generally Cooper, J. et at. The Biochemical Basis of Neuropharmacology, 161-195 (Oxford University Press, NY 3d Ed. 1978). The diverse physiological actions of dopumine are in mm mediated by its interaction with two of the basic types of G protein-coupled receptors: D1 and D2, which respectively stimulate and inhibit the enzyme adenylyl cyclase. Kebabian, J. and Calne, D., Nature 277:93-96 (1979). Alterations in the number or activity of these receptors may be a contributory factor in disease states such as Parkinson's disease (a movement disorder) and schizophrenia (a behavioral disorder).
A great deal of information has accumulated on the biochemistry of the D1 and D2 dopamine receptors, and methods have been developed to solubilize and purify these receptor proteins. See Senogles, S. et at., Biochemistry 25:749-753 (1986); Gingrich, J. et at., Biochemistry 27:3907-3912 (1988). The D1 dopamine receptor in several tissues appears to be a glycosylated membrane protein of about 72 kDa. Amlaiky, N. et at., Mol. Pharmacol. 31:129-134 (1987); Niznik, H. et al., Biochemistry 27:7594-7599 (1988). The D2 receptor has been suggested to have a higher molecular weight of about 90-150 kDa. Amlaiky, N. and Caron, M., J. Biol. Chem. 260:1983-1986 (1985); Amlaiky, N. and Caron, M., J. Neurochem. 47:196-204 (1986); Jarvie, J. et al., Mol. Pharmacol. 34:91-97 (1988). Much less is known about a recently discovered additional dopamine receptor, termed D3. Sokoloff, P. et al. Nature 347:146-151 (1990). Dopamine receptors are primary targets in the clinical treatment of psychomotor disorders such as Parkinson's disease and affective disorders such as schizophrenia. Seeman, P. et at, Neuropsychopharm. 1:5-15 (1987); Seeman, P. Synapse 1:152-333 (1987). The three different dopamine receptors (D1, D2, D3) have been cloned as a result of nucleotide sequence homology which exists between these receptor genes. Bunzow, J. R. et at. Nature 336:783-787 (1988); Grandy, D. K. el. al. Proc. Natl. Acad. Sci. U.S.A. 86:9762-9766 (1989); Dal Toso, R. et al. EMBO J. 8:40254034 (1989); Zhou, Q-Y. et at. Nature 346:76-80 (1990); Sunahara, R. K. et at. Nature 346:80-83 (1990); Sokoloff, P. et al. Nature 347, 146-151 (1990).
The antipsychotic clozapine is useful for socially withdrawn and treatment-resistant schizophrenics [Kane, J. et al. Nature 347:146-151 (1990)], but unlike other antipsychotic drugs, clozapine does not cause tardive dyskinesia [Casey, D. E. Psychophannacology 99: 547-553 (1989)]. Clozapine, however, has dissociation constants at D2 and D3 which are 3 to 30-fold higher than the therapeutic free concentration of clozapine in plasma water [Ackenheil, M. et al. Arzneim-Forsch 26:1156-1158 (1976); Sandoz Canada, Inc., Clozaril: Summary of preclinical and clinical data (1990)]. This suggests the existence of dopamine receptors more sensitive to the antipsychotic clozapine.
Van Tol et al., (1991) Nature, 350:610-614 report that they have cloned and sequenced a D2-like human dopamine receptor which they termed D4. They report that the dopamine D4 receptor gene has high homology to the human dopamine D2 and D3 receptor genes. They further report that the pharmacological profile of this receptor resembles that of the D2 and D3 receptors but that it has an affinity for clozapine which is tenfold higher. Van Tol et at. suggest that this characteristic may make the D4 receptor useful in discovering new types of drugs for schizophrenia that, like clozapine, do not induce tardive dyskinesia and other motor side effects.
Unfortunately, the anticipated low abundance of D4 receptors in the brain based on mRNA studies has hindered studies of the native D4 receptors. In addition, the genomic clone isolated and used by Van Tol et al. for expression contains introns. In a personal communication to the present inventors, H. H. M. Van Tol acknowledged that their expression construct could not be stably expressed. Therefore, a new approach is needed to facilitate the development of cell lines capable of the stable expression of a D4 dopamine receptor gene to further pharmacological and functional analysis of the D4 receptor.